The detection of specific DNA sequences by hybridization probes containing biotinylated nucleotide analogs, which are described in European Patent Application No. 0063 879A2 filed Apr. 6, 1982, is proving to be a very useful alternative to traditional procedures utilizing radiolabeled probes. Because of the extremely high affinity of the glycoprotein avidin for biotin (K.sub.dis =10.sup.-15 M), a detection system capable of detecting single copy mammalian DNA sequences has been developed based upon the formation of a ternary complex involving biotin, avidin and polymers of alkaline phosphatase. This highly sensitive detection system, coupled with other advantages of the biotin-avidin system such as probe stability (biotinylated DNA does not decay as does radiolabeled DNA) and the time required for detection (only 1 hr is required to visualize biotinylated DNA) promise to make the use of biotinylated DNA probes the method of choice in hybridization procedures in the very near future.
The highly specific and very strong binding of biotin to avidin (K.sub.D 10.sup.-15 M) is the major reason that the avidin-biotin interaction is also gaining in popularity as a tool for isolating macromolecules from crude physiological mixtures. The strong avidin-biotin complex can provide a one step, high yield retrieval of target macromolecules as biotinylated macromolecules from crude physiological mixtures. Unfortunately, once isolated, biotinylated macromolecules cannot be gently released from the avidin so that the target macromolecule can be obtained. Dissociation of the avidin-biotin complex usually requires 6 molar guanidine HCl, pH 1.5, an environment too extreme for many macromolecules.
The use of the guanido analog of biotin, 2-iminobiotin, does satisfy the problem of reversible biotinylation to some extent because avidin binds to 2-iminobiotin tightly at pH values of 9.5 and higher and dissociates at pH values of 4 and lower. However, in addition to the pH limitations associated with its use, the avidin-iminobiotin interaction is not nearly as strong as that of avidin-biotin.
There is a need for a gentle, nondestructive method for isolating target macromolecules from crude physiological mixtures. Such a method could be a useful tool in the identification, purification and subsequent characterization of a number of biologically significant macromolecules, such as DNA-protein complexes.